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Kontrasepsi Copper T (AKDR CuT) menggantikan kontrasepsi lippes loop (AKDR LL) disebabkan efektivitas yang lebih tinggi dan rendahnya angka ekspulsi. Kontrasepsi CuT diketahui mendukung kerusakan pada epitel endometrium dan menstimulasi produksi glikodelin A (GdA), yang memiliki peran bermakna dalam cara kerja kontrasepsi. Belum diketahui peran polietilen dalam meningkatkan GdA dan peran komponen inflamasi mengenai GdA.

 

Penelitian ini menggunalan desain penelitian eksplorasi yang dilakukan di Laboratorium Bagian Bedah Fakultas Kedokteran Hewan, Institut Pertanian Bogor pada November 2017–Maret 2018. Total 22 tikus sprague dawley yang dibagi menjadi kelompok polietilen + tembaga (AKDR CuT) dan kelompok polietilen tunggal (Kelompok LL AKDR). Darah dari arteri lakrimal dan jaringan dari kornua diperiksa dengan metode enzyme-linked immunosorbent assay (ELISA) dan imunohistokimia (IHK).

 

Sebagai hasilnya ditemukan kelompok kontrasepsi CuT menyebabkan perubahan lebih besar pada sel epitel permukaan endometrium dan sel-sel epitel kelenjar dibandingkan dengan kelompok kontrasepsi LL. Terdapat peningkatan yang bermakna pasca-penyisipan polietilen pada ekspresi TNF-α (p = 0,037) dan EGR-2 + (p = 0,039). Ditemukan peningkatan yang bermakna pasca-penyisipan polietilen pada ekspresi GdA (p < 0,001). Ekspresi delta GdA lebih besar pada polietilen 43,67 ± 36,36 dibandingkan kelompok CuT 13,50 ± 10,34.

 

Kesimpulan dari penelitian ini adalah ditemukan ekspresi GdA lebih besar dalam polietilen tunggal daripada kombinasi polietilen + tembaga (AKDR CuT). TNF-α dan makrofag CD38 + adalah komponen inflamasi terbesar yang menyebabkan peningkatan ekspresi GdA.

 

Kata kunci: glikodelin A, kerusakan epitel endometrium, komponen inflamasi, polietilen, polietielen+tembaga.

 

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Copper T IUD (CuT IUD) contraception replaces lippes loop IUD (LL IUD) due to its effectiveness and rarely expulsion capability. The CuT IUD is known to promote endometrial epithelial damage and to stimulate the production of glycodelin A (GdA). GdA has significant role in the contraception mechanism of action. It has not yet known the role of polyethylene in increasing GdA and the role of the inflammatory components regarding GdA.

 

This study is an exploratory research design conducted at the Laboratory of Surgery of the Faculty of Veterinary Medicine, Bogor Agricultural University on November 2017–March 2018. A Total of 22 sprague dawley rats were divided into polyethylene+copper  (CuT IUD) and single polyethylene (LL IUD groups). Blood from the lacrimal artery and tissue from the cornua were examined by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) methods.

 

It was found that CuT IUD group caused greater change to the endometrial surface epithelial cells and glandular epithelial cells compared to LL IUD group. There was a significant increase in post-insertion of polyethylene on the expression of TNF-α (p = 0.037) and EGR-2⁺ (p = 0.039). There was a significant increase in post-insertion of polyethylene on expression of GdA (p < 0,001). Delta expression of GdA was greater in the polyethylene 43.67 ± 36.36 than in the CuT IUD group 13.50 ± 10.34.

 

As a conclusion, the expression of GdA was found to be greater in the single polyethylene than combination of polyethylene+copper (CuT IUD). TNF-α and macrophages CD38⁺ are the biggest inflammatory components that cause an increase in GdA expression.

 

Keywords: endometrial epithelial damage, glycodelin A, inflammatory omponents, polyethylene, polyethylene+copper.

 

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"Latar belakang: Vitrifikasi folikel pre-antral menjadi pilihan dalam upaya preservasi fungsi reproduksi karena dapat menurunkan risiko mikrometastasis sel kanker akibat transplantasi korteks ovarium serta tidak dipengaruhi oleh perfusi jaringan. Tujuan: Memperoleh upaya preservasi fungsi ovarium yang efektif dengan penilaian apoptosis folikel pre-antral. Tempat: Departemen Obstetri Ginekologi Fakultas Kedokteran Universitas Indonesia - RS Dr. Cipto Mangunkusumo dan RS Fatmawati Jakarta. Metode: Studi ini merupakan penelitian eksperimental untuk menilai apoptosis folikel pre-antral pasca vitrifikasi. Folikel pre-antral segar merupakan kelompok kontrol. Hasil: Korteks ovarium didapatkan dari 6 enam pasien kanker serviks dan kanker payudara berumur 30-37 tahun yang menjalani operasi ooforektomi. Tidak terdapat perbedaan morfologi folikel primordial, folikel primer dan folikel sekunder dari korteks ovarium segar dan korteks ovarium pasca vitrifikasi. Dari 6 sampel korteks ovariumSeratus enam puluh satu berhasil di-isolasi 161 folikel pre-antral berhasil di-isolasi dengan 70 % di antaranya merupakan folikel sekunder. Tidak tampak perbedaan morfologi folikel pre-antral berdasarkan kriteria membran basalis, sel granulosa, zona pelusida dan oosit. Rerata ekspresi mRNAgen FasL folikel pre-antral segar adalah 0,43 ± 0,20 dibandingkan 0,51 ± 0,20 pada folikel pre-antral pasca vitrifikasi (nilai p = 0,22). Rerata ekspresi mRNAgen kaspase-3 folikel pre-antral segar adalah 0,56 ± 0,49 dibandingkan 0,27 ± 0,21 pada folikel pre-antral pasca vitrifikasi (nilai p = 0,233). Satu folikel sekunder dari korteks ovarium segar berhasil tumbuh menjadi folikel antral lanjut pada hari ke-6 kultur. Simpulan: Vitrifikasi folikel pre-antral terbukti tidak menyebabkan perubahan morfologi folikel dan peningkatan ekspresi gen mRNA FasL dan kaspase-3. Untuk membuktikan pengaruh vitrifikasi terhadap kesintasan folikel pre-antral pasca dalam kultur diperlukan penelitian lanjutan.

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Background: Pre-antral follicle vitrification should be considered as fertility preservation method because it lowers the risk of cancer micrometastasis of ovarian tissue transplantation and is not disturbed by ovarian tissue perfusion.

Objectives: To obtain the effective method of ovarian function preservation with pre-antral follicle apoptosis assessment.

Setting: Department of Obstetrics and Gynecology Faculty of Medicine Universitas Indonesia - Dr. Cipto Mangunkusumo General Hospital and Fatmawati Hospital Jakarta.

Method: This is an experimental study about apoptosis in pre-antral follicles after vitrification. Fresh pre-antral follicles served as a control group.

Results: Ovaries from six women between 30‒37 years of age who underwent oophorectomy due to cervical cancer or breast cancer were examined. There was no significant difference between primordial, primary and secondary follicles morphology from fresh and warmed-vitrified ovaries based on basal membrane, granulosa cells, zona pellucida and oocytes. From 6 six ovarian cortex, 161 pre- antral follicles were isolated and 70 % of them is secondary follicle. There was no significant difference between the morphology of isolated pre-antral follicles from fresh and warmed-vitrified ovaries. The mean FasL mRNA expression on the fresh isolated pre-antral follicles was 0.43±0.20 versus 0.51±0.20 on the warmed-vitrified group (p=0.22). The mean caspase-3 mRNA expression on the fresh isolated pre-antral follicles was 0.56±0.49 versus 0.27±0.21 on the warmed- vitrified group (=0.233). One secondary follicle grew and developed to an antral follicle within 6 days of culture.

Conclusion: It was shown that vitrification did not affect pre-antral follicles morphology and mRNA expression of FasL and caspase-3 on isolated pre-antral follicles and ovarian cortex. Further studies are required to establish whether vitrification affect in vitro culture of pre-antral follicles"

"Sindrom Ovarium Polikistik (SOPK) saat ini merupakan salah satu kelainan dengan keberhasilan kehamilan terendah di antara berbagai penyebab infertilitas. Penelitian ini bertujuan untuk menilai ultrasonografi (USG) sebagai prediktor diagnosis reseptivitas endometrium perempuan infertil Sindrom Ovarium Polikistik. Penelitian ini merupakan studi diagnostik observasional dengan disain potong lintang. Tiga puluh empat perempuan usia reproduksi (32,5 ± 3,8 tahun), mengalami infertilitas primer 4,9 ± 3,1 tahun dengan siklus anovulasi mendapat klomifen sitrat 100 mg perhari H2?6; perkembangan folikel dan ovulasi dikonfirmasi dengan pemeriksaan ultrasonografi (USG) H12?17. Pemeriksaan USG yang diikuti biopsi endometrium dan hormon progesteron dilakukan pada H19?21 atau pasca ovulasi H+5?+7. USG digunakan untuk menilai Zona Vaskularisasi menurut kriteria Sonai, Volume Endometrium menurut kriteria Zollner, dan Indeks Vaskularisasi-Arus Darah menurut kriteria Wu. Biopsi endometrium dinilai berdasarkan penanggalan histopatologis menurut kriteria Noyes, dan pemeriksaan imunohistokimia VEGF dan VEGFR-1 dengan penilaian secara H-Score. Kadar VEGF serum diperiksa dengan metode Elisa. Analisis statistik menggunakan uji chi-square, uji-t dan nilai ROC. Dihasilkan titik potong komposit endometrium sebagai baku emas reseptivitas endometrium berdasarkan pemeriksaan penanggalan histopatologis endometrium. Pemeriksaan USG berdasarkan pemeriksaan komposit endometrium ini akhirnya menghasilkan baku emas USG penetapan reseptivitas endometrium. Pemeriksaan USG H19?21 menunjukkan rerata tebal endometrium 10,47 ± 1,85 mm, Volume Endometrium 3,70 ± 1,31 ml, Indeks Vaskularisasi?Arus Darah Indeks Vaskularisasi?Arus Darah 0,08 (0,00-3,21) dan Zona Vaskularisasi di lapis 1,2,3 dan 4 masing-masing 14,7%, 41,2%, 35,3% dan 8,8%. Pemeriksaan histopatologis endometrium mendapatkan 58,8% in-phase dan 41,2% out-phase. Pemeriksaan VEGF endometrium mendapatkan ekspresi tertinggi di endotel (2,34 ± 0,26), kemudian di epitel luminal (2,23 ± 0,37), sel stroma (2,1±1,9), terendah di epitel kelenjar (2,00 ± 0,68). VEGFR-1 endometrium tertinggi di epitel kelenjar (2,85 ± 0,30), diikuti di epitel luminal (2,83 ± 0,54), endotel (2,70 ± 0,42) dan terendah di sel stroma (2,58 ± 0,42). Secara statistik, ditemukan hubungan bermakna antara Zona Vaskularisasi dengan VEGF sel stroma (p = 0,018), Volume Endometrium dengan VEGF endotel (p = 0,000), epitel luminal (p = 0,029) dan total sel (0,043) serta Penanggalan Histologis Endometrium dengan VEGFR-1 sel stroma (p = 0,009). Penetapan reseptivitas endometrium hasil penilaian Komposit USG berdasarkan baku emas komposit endometrium adalah ditemukannya Zona Vaskularisasi lapis 3?4, Volume Endometrium ≥ 3,090 ml dan Indeks Vaskularisasi-Arus Darah ≥ 0,253 yang menunjukkan spesifisitas 77,4%. Ultrasonografi dapat digunakan sebagai prediktor diagnosis reseptivitas endometrium masa jendela implantasi embrio perempuan infertil SOPK.

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Polycyctic ovary syndrome has been recognized as one of the lowest successful pregnancy rates in infertile women. This studi aimed to assess ultrasound as predictor of endometrial receptivity in PCOS infertile women.

Diagnostic observational study in cross sectional design was conducted. Thirty-four subjects suffered anovulatory cycles in a average 32,5 ± 3,8 years of age and primary infertility for 4,9 ± 3,1 years, receiving 100 mg/d clomiphene citrate therapy on D2?6 . Follicular development and ovulation were confirmed by tranasvaginal USG examination on D12?17 . Repeated USG procedures followed by endometrial biopsy and serum progesterone test were conducted on either D 19?21 or D+5?+7 post ovulatory. The use of USG was to assess Vascularization Zone by Sonai criteria, Endometrial Volume by Zollner criteria and Vascularization Flow Index (VFI) by Wu criteria. Endometrial biopsy was performed and dated, based on endometrial histological dating by Noyes citeria. Immunohistochemistry of VEGF and VEGFR-1 were done and counted by H-Score formula. VEGF serum was tested by Elisa method. Statistical analysis of Chi-squre test, student t-test and ROC value were used. Immunohistochemistry composite formation was based on histological dating of endometrium. Ultrasound composite based on immunohistochemistry composite was finally resulting the new cut-off of endometrial reseptivity. Ultrasound findings on D19?21 showed the average endometrial thickness 10,47± 1,85 mm, Endometrium Volume 3,70 ± 1,31 ml, Vascularization?Flow Index (VFI) 0,08 (0,00?3,21 ) and Vascularization Zone (ZV) of zone 1,2,3 and 4 were 14,7%, 41,2%, 35,3% and 8,8%. Endometrial dating was 58,8% in-phase and 41,2% out-phase. Endometrial VEGF staining showed the highest expression in endothel (2,34 ± 0,26), followed by luminal epithelium (2,23 ± 0,37), stromal cells (2,1 ± 1,9) and the lowest in glandular epithelial (2,00 ± 0,68); meanwhile the highest VEGFR-1 expression was seen in glandular epithelial (2,85 ± 0,30), followed by luminal epithelial (2,83 ± 0,54), endothelial (2,70 ± 0,42) and the lowest at the stromal cells (2,58 ± 0,42). Statistically, ZV was correlated to the VEGF stromal cells (p = 0,018) and Endometrial Volume was correlated to VEGF endothelial (p = 0,000) and VEGF luminal epithelium (p = 0,029) and VEGF total cells (p = 0,043); meanwhile Histological Dating of Endometrium was correlated to VEGFR-1 stromal cells (p = 0,009). Endometrial receptivity predictor determined by Ultrasound Composite based on immunohistochemistry composite was Vascularization Zone of layer 3?4, Endometrial Volume of ≥ 3,090 ml and endometrial VFI of 0,253 with a specificity of 77,4%. Ultrasound was the useful tools for diagnostic predictor of endometrial receptivity diagnosis during the implantation windows period of PCOS infertile female."

"Latar Belakang: DNA bebas dalam medium kultur embrio dan kemajuan pemodelan berbasis kecerdasan buatan berpotensi menjadi modalitas uji genetik yang non-invasif. Saat ini, tidak diketahui apakah DNA tersebut dilepaskan oleh sel embrio euploid atau aneuploid, sehingga melemahkan dasar keilmuan penggunaannya. Penelitian ini bertujuan untuk mengetahui sel sumber embrio pelepas DNA bebas dalam medium kultur, validasi potensi klinis penggunaan DNA bebas untuk skrining status ploidi embrio pasien Fertilisasi In-Vitro (FIV), dan konstruksi model pembelajaran mendalam menggunakan gambar embrio tersegmentasi untuk deteksi status ploidi embrio.Metode: Penelitian ini terbagi dalam dua desain penelitian yaitu eksperimental in-vitro menggunakan embrio hewan model dan observasi kohort menggunakan 28 sampel medium kultur embrio dari 21 pasien program FIV di Klinik Morula IVF Jakarta, periode September 2022–Januari 2023. Konstruksi model pembelajaran mendalam menggunakan gambar embrio pasien FIV yang menjalani program bayi tabung periode Januari 2021– Juni 2023. Deteksi sel embrio sumber pelepas DNA bebas dilakukan dengan memapar salah satu embrio (galur DDY atau C57BL) dengan reversin untuk memperoleh blastomer pembawa sel-sel aneuploid. Embrio kontrol dikultur bersamaan tanpa reversin sebagai pembawa sel-sel blastomer euploid. Agregasi membentuk embrio mosaik dilakukan antara embrio pembawa blastomer aneuploid (perlakuan) dan embrio pembawa blastomer euploid (kontrol). Polimorfisme gen GABRA2 antara galur DDY (alel wildtype) dan C57BL (alel delesi) mejadi alel target yang dikuantifikasi dengan metode qPCR. Empat jenis sampel dibuat sebagai berikut: medium kultur tanpa embrio, medium kultur embrio agregasi tanpa pemaparan reversin, rev-DDY, rev-C57BL. Embrio mosaik diwarnai dengan marka apoptosis untuk deteksi mekanisme pelepasan DNA bebas. Analisis status ploidi embrio menggunakan medium kultur embrio pasien FIV dilakukan dengan metode sekuensing. Konstruksi model pembelajaran mendalam menggunakan gambar embrio tersegmentasi yang dipotong urut selama 10 jam sebelum proses biopsi. Variabel yang diamati dalam penelitian adalah konsentrasi alel delesi dan wildtype gen GABRA2, jumlah sel terwarnai marka apoptosis, Pada sampel medium kultur embrio manusia, keberhasilan amplifikasi dan interpretasi hasil sekuensing, serta tingkat kesesuaian uji antara DNA bebas dengan biopsi trofoblas dianalisis. Kemampuan prediksi model berbasis kecerdasan buatan dinilai dengan akurasi dan loss. Analisis data penelitian dan konstruksi model pembelajaran mendalam menggunakan perangkat lunak SPPS versi 21, OpenEpi. pyhton.Hasil: Sebanyak 0,08 ng/reaksi alel wildtype ditemukan pada embrio mosaik dengan pemaparan reversin pada blastomer embrio DDY (rev-DDY, pembawa sel-sel aneuploid) dan 0,01 ng/reaksi alel delesi ditemukan pada embrio mosaik dengan pemaparan reversin pada blastomer embrio C57BL (rev-C57BL, pembawa sel-sel aneuploid). Median jumlah sel embrio terwarnai marka apoptosis antara ketiga group embrio (agregasi kontrol, rev- DDY dan rev-C57BL) tidak berbeda bermakna (nilai p = 0,95 untuk pewarnaan late apoptosis (propidium iodide) dan p = 0,42 untuk early apoptosis (Ann-V) menandakan adanya proses koreksi sel pada kedua group embrio mosaik selama masa perkembangan pra-implantasi. Keberhasilan amplifikasi DNA bebas medium kultur embrio manusia dalah 100%, dengan nilai interpretasi 92,8% (26/28). Nilai kesesuaian DNA bebas dengan biopsi trofoblas adalah rendah sebesar 65,4% (17/26) dengan kesesuaian kromosom seks adalah 61,5% (16/26). Sepuluh dari 11 embrio XY pada biopsi trofoblas terdeteksi XX pada DNA bebas. Seluruh model pembelajaran mendalam mengalami peningkatan akurasi menggunakan gambar embrio tersegmentasi dengan algoritma InceptionV3 mencapai akurasi tertinggi sebesar 0,67 dengan nilai loss sebesar 1,4.Kesimpulan: Sel embrio anueploid adalah sel sember pelepas DNA bebas medium kultur embrio pada embrio mosaik hewan coba mencit yang dilepaskan melalui mekanisme apoptosis. Embrio masik tersebut diperkirakan melakukan self-correction dengan mengeksklusi sel-sel aneploid untuk mempertahankan euploiditasnya. Rendahnya tingkat kesesuaian antara DNA bebas dengan biopsi trofoblas disebabbkan oleh adanya kontaminasi maternal yang ditandai dengan perubahaan koromosm seks yang signifikan. Penggunaan gambar blastosis tersegmentasi meningkatkan akurasi model prediksi pembelajaran mendalam.

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Background: Cell-free DNA and advanced artificial intelligence-based modeling uphold the potential of a non-invasive approach to determining embryo ploidy status. The specific embryonic cells (whether euploid or aneuploid) that release cell-free DNA are largely unknown, causing a weak scientific basis for its use. This study aimed to identify the source of embryonic cells releasing cell-free DNA in culture media, validate the clinical potential of using cell-free DNA to screen embryo ploidy status in an in-vitro fertilization (IVF) program and develop a deep learning model using segmented embryo images to detect embryo ploidy status.

Materials and Methods: This study employed two research designs including an in-vitro experimental study using animal model embryos and an observational cohort study using 28 samples of spent embryo culture media from 21 patients undergoing IVF program at Morula IVF Clinic Jakarta (September 2022 to January 2023). A deep learning model was constructed using images of embryos from IVF patients who participated in IVF program from January 2021 to June 2023. Detection of the source embryonic cells releasing cell-free DNA was achieved by exposing embryos (DDY or C57BL strains) to reversine to induce the formation of blastomeres carrying aneuploid cells. Control embryos were cultured simultaneously without reversine to serve as the source of euploid blastomeres. Mosaic embryo aggregation was performed by combining embryos carrying aneuploid blastomeres (treatment) with those carrying euploid blastomeres (control). The GABRA2 gene polymorphism between the DDY strain (wildtype allele) and the C57BL strain (deletion allele) was the target allele quantified using qPCR. Four types of samples were prepared: culture medium without embryos, culture medium of aggregated embryos without reversine exposure, rev-DDY, and rev-C57BL. Mosaic embryos were stained with an apoptosis marker to detect the mechanism of cell-free DNA release. The ploidy status of embryos using spent embryo culture media from IVF patients was determined using sequencing methods. The deep learning model was constructed using segmented images of embryos captured over 10 hours before the biopsy process. The variables observed in the study included the concentration of deletion and wildtype alleles of the GABRA2 gene, the number of cells stained with apoptosis markers, the success rate of amplification and interpretation of sequencing results from human spent embryo culture medium samples, and the concordance rate between cell-free DNA and trophectoderm biopsy analysis. The predictive ability of the artificial intelligence-based model was evaluated using accuracy and loss metrics. Data analysis and deep learning model construction were performed using SPSS version 21, OpenEpi, and Python.

Results: A total of 0.08 ng/reaction of the wildtype allele was detected in the culture media sample of mosaic embryos exposed to reversine in DDY embryo blastomeres (rev- DDY, carrying aneuploid cells), and 0.01 ng/reaction of the deletion allele was found in the sample exposed to reversine in C57BL embryo blastomeres (rev-C57BL, carrying aneuploid cells). The median number of embryonic cells stained with apoptosis markers among the three groups of embryos (control aggregation, rev-DDY, and rev-C57BL) did not differ significantly (p = 0.95 for late apoptosis staining with propidium iodide and p = 0.42 for early apoptosis with Annexin V), indicating the presence of cell correction processes in both groups of mosaic embryos during pre-implantation development. The success rate of cell-free DNA amplification in human spent embryo culture media was 100%, with an interpretability of 92.8% (26/28). The concordance between cell-free DNA and trophectoderm biopsy was low at 65.4% (17/26), with sex chromosome concordance at 61.5% (16/26). Ten out of eleven XY embryos from the trophectoderm biopsy were detected as XX in cell-free DNA analysis. All deep learning models showed improved accuracy using segmented embryo images with the InceptionV3 algorithm, achieving the highest accuracy of 0.67 with a loss of 1.4.

Conclusion: Aneuploid embryonic cells were identified as the source releasing cell-free DNA in culture media during embryo animal model experiments, releasing DNA through an apoptotic mechanism. These mosaic embryos were expected to activate embryonic cell correction mechanisms by excluding aneuploid cells to maintain their euploidy. The low concordance rate between cell-free DNA and trophectoderm biopsy was attributed to maternal contamination, as indicated by significant changes in sex chromosomes. The use of segmented blastocyst images improved the model's accuracy.

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